相关实验视频

Updated: Jun 6, 2025

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Published on: September 16, 2019

提高了有序双模板继电器cDNA库准备用于RNA测序的精度,灵敏度和适应性

Lucas Ferguson^1,2, Heather E Upton^1,3, Sydney C Pimentel^1,4

¹Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, USA.

bioRxiv : the preprint server for biology

|November 22, 2024

概括

优化的有序双模板中继 (OTTR) 方法提高了RNA测序精度和小RNA的产量. 这种增强的协议最大限度地减少了偏差和细菌污染,从而实现了精确的端到端RNA序列捕获.

科学领域:

分子生物学分子生物学
基因组学就是基因组学.
生物技术是生物技术.

背景情况:

传统的小RNA测序方法由于涉及结合和多核酸尾部的多步骤协议,因此产量低,偏差和信息丢失.
订购的两模板继电器 (OTTR) 之前被开发用于端到端的RNA序列捕获和适配器附加在一个单一的步骤中,对微RNA,tRNA和核糖体保护的mRNA足迹库进行了基准测试.

研究的目的:

描述,量化和减少OTTR端到端RNA序列捕获中的偏差和不准确性.
为了优化反应缓冲区,逆转录酶,适配器寡核酸和整体工作流程,以改进RNA库准备.

主要方法:

开发和应用新的指标来评估RNA序列捕获效率和精度.
逆转录酶和适配器寡核酸的优化,以提高3'和5'末端精度.
改善重组表达和净化截断的Bombyx mori R2逆转录酶,以尽量减少细菌核酸污染.
引入一个快速的,自动化兼容的,无凝的cDNA丰富协议.

主要成果:

修改后的逆转录酶和适配器寡核酸显著提高了RNA序列捕获的3'和5'终端精度,并将图书馆偏差降到最低.
增强R2逆转录酶的净化减少了非生产性测序读数,细菌核酸污染物<10%,即使有3皮克克的miRNA输入.
新的协议使cDNA复合体的无凝,无长度的丰富成为可能,有效地将它们与仅适配器产品分开.

结论:

精细的OTTR协议为任何序列,结构或修改的RNA提供了改进的无偏见,端到端的RNA序列捕获和注释.
这种优化的方法提高了小RNA测序的可靠性和效率,特别是对于低输入样本.
这些进步为全面的RNA分析提供了一个强大的平台,解决了以前测序协议的局限性.

关键词:

这就是OTTR OTTR.没有编码的RNA.没有模板的核酸添加.反向转录酶的使用模板跳跃的模板跳跃终端转移的情况

更多相关视频

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

Published on: November 18, 2014

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Author Spotlight: AQRNA-seq Role in Mapping Small RNAs and Unraveling Protein Translation Mechanisms

Published on: February 2, 2024

相关概念视频

RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

RACE - Rapid Amplification of cDNA Ends

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific...